PROJECT SUMMARY Substance Use Disorder (SUD) remains one of the most difficult to treat of all neuropsychiatric disorders, largely due to the high propensity for relapse, even after prolonged periods of abstinence, owing to craving following re- exposure of addicts to drug-conditioned stimuli. Cocaine-addicted patients, in particular, maintain relatively high relapse rates; yet effective treatments for relapse in clinical populations remain elusive. Accordingly, the need for a better understanding of the biology underlying cue-induced relapse remains critical for designing effective treatments for addiction. Clinical neuroimaging experiments show heightened activation of cortical and downstream limbic regions, including the prefrontal cortex, amygdala, and nucleus accumbens, in addicts exposed to cocaine-conditioned cues (i.e. paraphernalia). Cocaine self-administration studies in rats reveal that heightened glutamate transmission in the nucleus accumbens core (NAcore), and the subsequent activation of a sparse population of neuronal nitric oxide synthase (nNOS)-expressing interneurons, regulates cue-induced craving for cocaine. However, simultaneous, near real time, measurements of glutamate and nitric oxide (NO) concentrations in the NAcore during cue-induced cocaine seeking has not been performed. Moreover, it is unknown what glutamate inputs, and what glutamate receptors on nNOS interneurons, are required for cue- induced NO release. The major goal of this proposal is to disentangle the glutamate inputs to the NAcore, and glutamate receptor subtypes on nNOS interneurons, required for cue-induced increases in extracellular glutamate and NO in the NAcore, respectively. This will be addressed using the state-of-the-art techniques described herein. This goal will be addressed through two specific aims. The first aim is to determine whether cue-induced activation of prelimbic (PL) cortical (Aim 1a) or basolateral amygdala (BLA, Aim 1b) neurons projecting to the NAcore is required for cue-induced glutamate release, subsequent NO release, and seeking. To address this aim, we will use a biosensor-based recording strategy in freely moving rats; allowing for near real time detection of glutamate and NO levels in the NAcore during cue-induced reinstatement. This will be combined with transient inhibition of these two glutamate inputs to the NAcore to examine the relative contribution of each to cue-induced glutamate and NO release. The second aim will be to determine whether mGluR5 (Aim 2a) or GluN2B (Aim 2b) receptors on nNOS interneurons mediates cue-induced NO release in the NAcore and cocaine seeking. This aim will be addressed using the same recording strategy described above combined with Cre-dependent viral vectors driving shRNA against these two receptors in male and female mice expressing temporally regulated nuclear Cre in nNOS interneurons. These studies will fill a critical role in our understanding of the neurochemical mechanisms underlying relapse; delineating the involvement of discrete glutamate inputs and receptor systems on nNOS interneurons responsible for seeking. The training required to accomplish these aims will undoubtedly facilitate my career goal of becoming an independent academic neuroscientist.